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• W2005784265 abstract "Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) α-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, β-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Δp) was 15% lower than controls. The change affected the ΔΨ component only, leaving the ΔpH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARα-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARδ, we discuss how this subtype might compensate for deficiency of PPARα. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others. Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) α-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, β-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Δp) was 15% lower than controls. The change affected the ΔΨ component only, leaving the ΔpH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARα-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARδ, we discuss how this subtype might compensate for deficiency of PPARα. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others. Administration of 3-thia fatty acids to rats leads to hypolipidemia. The metabolism and biological effects of these non-oxidizable fatty acid analogues, of which tetradecylthioacetic acid (TTA) 1The abbreviations used are: TTA, tetradecylthioacetic acid; Δp, proton electrochemical potential; ΔΨ, membrane potential (electrical potential difference); ΔpH, pH difference; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; PPAR, peroxisome proliferator-activated receptor; UCP, uncoupling protein; TTP, tetraphenylphosphonium; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.1The abbreviations used are: TTA, tetradecylthioacetic acid; Δp, proton electrochemical potential; ΔΨ, membrane potential (electrical potential difference); ΔpH, pH difference; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; PPAR, peroxisome proliferator-activated receptor; UCP, uncoupling protein; TTP, tetraphenylphosphonium; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. is the most studied, have been reviewed (1Berge R.K. Hvattum E. Pharmacol. Ther. 1994; 61: 345-383Crossref PubMed Scopus (40) Google Scholar, 2Skrede S. Sorensen H.N. Larsen L.N. Steineger H.H. Hovik K. Spydevold O.S. Horn R. Bremer J. Biochim. Biophys. Acta. 1997; 1344: 115-131Crossref PubMed Scopus (80) Google Scholar, 3Bremer J. Prog. Lipid Res. 2001; 40: 231-268Crossref PubMed Scopus (59) Google Scholar, 4Berge R.K. Skorve J. Tronstad K.J. Berge K. Gudbrandsen O.A. Grav H. Curr. Opin. Lipidol. 2002; 13: 295-304Crossref PubMed Scopus (61) Google Scholar). A considerable body of evidence points to shifts in the liver cellular metabolism, resulting in channeling of fatty acids to an enhanced mitochondrial β-oxidation, at the expense of triacylglyceol synthesis. Simultaneously, there is up-regulation of the inner carnitine palmitoyltransferase II, 2,4-dienoyl-CoA reductase, and mitochondrial 3-hydroxy-3-methyl-CoA synthase. The outer carnitine palmitoyltransferase-I is not affected, suggesting that the rate control of β-oxidation and ketogenesis resides in steps beyond acyl group translocation into the matrix (4Berge R.K. Skorve J. Tronstad K.J. Berge K. Gudbrandsen O.A. Grav H. Curr. Opin. Lipidol. 2002; 13: 295-304Crossref PubMed Scopus (61) Google Scholar, 5Willumsen N. Vaagenes H. Rustan A.C. Grav H. Lundquist M. Skattebol L. Songstad J. Berge R.K. J. Lipid Mediat. Cell Signal. 1997; 17: 115-134Crossref PubMed Scopus (7) Google Scholar, 6Madsen L. Garras A. Asins G. Serra D. Hegardt F.G. Berge R.K. Biochem. Pharmacol. 1999; 57: 1011-1019Crossref PubMed Scopus (27) Google Scholar). Modulation of lipid metabolism with TTA seems at least in part to be related to the role of TTA as a regulator for members of the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. TTA has been demonstrated to function as a ligand and activator of the PPAR subtypes PPARα, PPARδ, and PPARγ (4Berge R.K. Skorve J. Tronstad K.J. Berge K. Gudbrandsen O.A. Grav H. Curr. Opin. Lipidol. 2002; 13: 295-304Crossref PubMed Scopus (61) Google Scholar, 7Raspe E. Madsen L. Lefebvre A.M. Leitersdorf I. Gelman L. Peinado-Onsurbe J. Dallongeville J. Fruchart J.C. Berge R. Staels B. J. Lipid Res. 1999; 40: 2099-2110Abstract Full Text Full Text PDF PubMed Google Scholar, 8Berge K. Tronstad K.J. Flindt E.N. Rasmussen T.H. Madsen L. Kristiansen K. Berge R.K. Carcinogenesis. 2001; 22: 1747-1755Crossref PubMed Scopus (58) Google Scholar, 9Madsen L. Guerre-Millo M. Flindt E.N. Berge K. Tronstad K.J. Bergene E. Sebokova E. Rustan A.C. Jensen J. Mandrup S. Kristiansen K. Klimes I. Staels B. Berge R.K. J. Lipid Res. 2002; 43: 742-750Abstract Full Text Full Text PDF PubMed Google Scholar). PPARα is the predominant subtype in the liver where it controls transcription of genes involved in fatty acid metabolism, such as the genes for peroxisomal acyl-CoA oxidase and fatty acid transport protein, which are up-regulated after TTA treatment (7Raspe E. Madsen L. Lefebvre A.M. Leitersdorf I. Gelman L. Peinado-Onsurbe J. Dallongeville J. Fruchart J.C. Berge R. Staels B. J. Lipid Res. 1999; 40: 2099-2110Abstract Full Text Full Text PDF PubMed Google Scholar, 9Madsen L. Guerre-Millo M. Flindt E.N. Berge K. Tronstad K.J. Bergene E. Sebokova E. Rustan A.C. Jensen J. Mandrup S. Kristiansen K. Klimes I. Staels B. Berge R.K. J. Lipid Res. 2002; 43: 742-750Abstract Full Text Full Text PDF PubMed Google Scholar). Mitochondrial uncoupling by fatty acids has been widely demonstrated during the last decades. Energy coupling is impaired when protons and other ions are allowed to pass through the inner membrane without the production of ATP. Consequently, the stored energy from the mitochondrial proton gradient intended for ATP synthesis is converted to heat. Wojtczak et al. (10Wojtczak L. Wieckowski M.R. Schonfeld P. Arch. Biochem. Biophys. 1998; 357: 76-84Crossref PubMed Scopus (59) Google Scholar) have demonstrated protonophoric behavior in vitro of high concentrations of 3-thia fatty acids toward the mitochondrial inner membrane. The concentration range causing rapid transbilayer movement of acyl chains was on par with that of normal, unipolar long chain fatty acids like palmitic or oleic acids. A similar behavior has been shown to apply to other hypolipidemic fatty acid analogues, such as β,β′-methyl-substitutedhexadecane-α,ω-dioic acid (11Hermesh O. Kalderon B. Bar-Tana J. J. Biol. Chem. 1998; 273: 3937-3942Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The molecular basis for fatty acid-mediated uncoupling of respiration remains unclear, but both passive and protein-mediated mechanisms appear to be involved. Skulachev (12Skulachev V.P. FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (392) Google Scholar) introduced the hypothesis of fatty acid cycling, assuming spontaneous translocation (flip-flop) of the protonated form of the fatty acid in one direction (toward matrix) and a transfer of the anionic form in the other direction, mediated by some inner membrane proteins. Putative candidates for such proteins are the ADP/ATP antiporter and the uncoupling proteins (UCPs) (12Skulachev V.P. FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (392) Google Scholar, 13Jezek P. Engstova H. Zackova M. Vercesi A.E. Costa A.D. Arruda P. Garlid K.D. Biochim. Biophys. Acta. 1998; 1365: 319-327Crossref PubMed Scopus (184) Google Scholar). UCP homologues form a family of mitochondrial carriers that are capable of depleting the proton gradient. The UCP subtypes, UCP-1, UCP-2, and UCP-3, differ in respect to tissue distribution and probably also function. UCP-1 appears to be solely expressed in brown adipose tissue where it mediates thermogenesis, whereas UCP-2 and UCP-3 are more widely expressed. The functions of UCP-2 and UCP-3 are still unclear, but a mild uncoupling of respiration could prevent the accumulation of oxygen radicals and/or control the NAD+/NADH ratio and consequently regulate metabolic pathways such as ketogenesis and lipogenesis (14Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (806) Google Scholar, 15Ricquier D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (745) Google Scholar). The activities of the UCPs are induced by free fatty acids (16Jacobsen S.E. Fahlman C. Blomhoff H.K. Okkenhaug C. Rusten L.S. Smeland E.B. J. Exp. Med. 1994; 179: 1665-1670Crossref PubMed Scopus (31) Google Scholar). Furthermore, mono- and polyunsaturated fatty acids, but not saturated fatty acids, were found to increase UCP-2 expression in hepatocytes possibly via a PPARα-mediated pathway (17Armstrong M.B. Towle H.C. Am. J. Physiol. 2001; 281: E1197-E1204PubMed Google Scholar). Others have found that PPARα mediates in vivo regulation of hepatic ucp-2 gene expression and that PPARγ has the same property in brown adipose tissue (18Kelly L.J. Vicario P.P. Thompson G.M. Candelore M.R. Doebber T.W. Ventre J. Wu M.S. Meurer R. Forrest M.J. Conner M.W. Cascieri M.A. Moller D.E. Endocrinology. 1998; 139: 4920-4927Crossref PubMed Scopus (256) Google Scholar). These observations suggest the possibility that PPAR activation and increased β-oxidation rate in liver mitochondria of rats fed TTA might be associated with increased proton conductance across the membrane. We have investigated whether this occurs in vivo after long term feeding of TTA to rats, by measuring energy state parameters at the tissue level, the cellular level, as well as at the level of isolated mitochondria, and if so to assess the extent to which such a mechanism might contribute to increased fatty acid oxidation. Materials—TTA was synthesized as described previously (19Madsen L. Froyland L. Grav H.J. Berge R.K. J. Lipid Res. 1997; 38: 554-563Abstract Full Text PDF PubMed Google Scholar). 3H2O, [U-14C]sucrose, [1-14C]palmitoyl-l-carnitine, [1-14C]palmitoyl-CoA, [3H]inulin, [3H]tetraphenylphosphonium (TTP), [14C]5,5′-dimethyloxazoline-2,4-dione, and dextran T-40 were obtained from Amersham Biosciences. Unlabeled species of the same compounds were purchased from Sigma. Saponin was from Fluka Chemica-Biochemica, Switzerland. Other chemicals were of the highest purity commercially available. Animals—Male Wistar rats were obtained from Møllegaard Breeding Laboratory, Eiby, Denmark. They were housed in pairs in wire cages and maintained on a 12-h cycle of light and dark at 20 ± 3 °C. The rats had free access to pellet food and water, and they were acclimatized to these conditions for at least 1 week prior to the experiment. Each test and control group consisted of at least 4 animals. If not otherwise stated, palmitic acid and TTA were separately dissolved in acetone and sprayed on pellets to an amount of 3 g/kg of pellets, resulting in an approximate daily dose of 300 mg/kg body weight (estimated consumption of pellets per rat, 20 g; body weight near 200 g). At day 7, the rats were subjected to a 12-h fast before termination. The PPAR–/– and PPAR+/+ mice (20–25 g) were a generous gift from Frank J. Gonzales (National Cancer Institute, Bethesda, MD) and have been described elsewhere (20Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1487) Google Scholar). The PPAR–/– and PPAR+/+ lines were purebred on a sv129 background. The mice were given a diet consisting of 21.8% casein, 10% soy oil, 17% vitamin/mineral mixture (5.83% vitamin mixture, AIN-93VX, Dyets Inc.; 17.4% mineral mixture, AIN-93G, Dyets Inc.; 11.6% cellulose; 63,9% sucrose; 1.2% cholintartrate) and dextrin (49.5–51.2%), supplemented with 0.5% fenofibrate (gift from Alan Edgar, Fournier, France) or 1.7% TTA. At termination the animals were anesthetized with a subcutaneous injection of Hypnorm Dormicum™ (fentanyl/fluanisone midazol-am, 0.2 ml/100 g body weight). Livers were either immediately removed, placed in ice-cold homogenizing medium and weighed, or freeze-clamped in situ and stored in liquid N2. Cardiac puncture was performed to obtain blood samples in EDTA vacutainers; one aliquot being frozen in liquid N2 for later measurement of blood nucleotides. The Norwegian State Board of Biological Experiments with Living Animals approved the protocol. Extraction and Measurement of Nucleotides—Nucleotides, including NAD+ and NADP+, were extracted with perchloric acid, whereas pyridine nucleotides in reduced form were obtained by alkaline extraction according to Williamson and Corkey (21Williamson J.R. Corkey B.E. Methods Enzymol. 1979; 55: 200-222Crossref PubMed Scopus (123) Google Scholar). Extracted nucleotides were separated, identified, and quantified on an ion pair reversed-phase high-performance liquid chromatographic system (22Stocchi V. Cucchiarini L. Canestrari F. Piacentini M.P. Fornaini G. Anal. Biochem. 1987; 167: 181-190Crossref PubMed Scopus (193) Google Scholar). Inorganic phosphate was determined by an assay based on the production of phosphomolybdate, which can be measured photometrically at 340 nm (Bayer AG, Leverkusen, Germany). The liver contents of each component were corrected for the amounts due to blood contaminating the tissue as described by Hohorst et al. (23Hohorst H.J. Kreutz F.H. Bücher T. Biochem. Z. 1959; 332: 18-46PubMed Google Scholar), by measurement of the oxyhemoglobin concentration, in the presence of saponin, of blood and liver tissue. Tissue energy parameters were calculated as follows: energy charge = ½{([ADP] + 2[ATP])/([AMP]+[ADP]+[ATP])} and phosphorylation state = [ATP]/[ADP][Pi] (nanomoles/g of tissue)–1 (see Ref. 24Krebs H.A. Veech R.L. Papa S. Tager J.M. Quagliariello E. Slater E.C. The Energy Level and Metabolic Control of Mitochondria. Ariatica Editrice, Bari, Italy1969: 329-382Google Scholar). Isolation of Mitochondria and Measurements of Enzyme Activities— The livers of individual animals were homogenized in an ice-cold medium consisting of 0.25 m sucrose, 10 mm HEPES buffer, pH 7.4, 1 mm EGTA. The mitochondrial fraction was obtained by differential centrifugation as described earlier (25Berge R.K. Flatmark T. Osmundsen H. Eur. J. Biochem. 1984; 141: 637-644Crossref PubMed Scopus (112) Google Scholar) at 0–4 °C. The Bio-Rad protein kit (Bio-Rad) was used for protein determination with bovine serum albumin (Sigma) as standard. Until used the mitochondrial fractions were stored at 0 °C at a concentration of 100 mg of protein/ml. Rates of oxygen consumption were measured polarographically as described earlier (26Grav H.J. Pedersen J.I. Christiansen E.N. Eur. J. Biochem. 1970; 12: 11-23Crossref PubMed Scopus (70) Google Scholar), the system being calibrated with air-saturated distilled water. Measurements were made at 25.0 °C, and where not specified otherwise the medium contained the following components at the indicated concentrations: 83 mm KCl, 4 mm KH2PO4,20mm K-HEPES buffer, pH 7.4, 1 mm EGTA, 1 mm MgCl2, and 4 mg of mitochondrial protein, in a total volume of 1.0 ml. Substrates were added as follows: 240 μm palmitoyl-l-carnitine + 5 mm malate (or 0.5 mm malate as indicated), 210 μm pamlitoyl-CoA + 1 mm l-carnitine + 5 mm malate (or 0.5 mm malate as indicated), or 5 mm sodium succinate + 1 μg of rotenone. Where indicated 40 μm FCCP was added near the end of the experiment. Rates of β-oxidation were determined by incubating mitochondria at 30 °C with [1-14C]palmitoyl-l-carnitine or [1-14C]palmitoyl-CoA + 1 mm l-carnitine, as described in Ref. 6Madsen L. Garras A. Asins G. Serra D. Hegardt F.G. Berge R.K. Biochem. Pharmacol. 1999; 57: 1011-1019Crossref PubMed Scopus (27) Google Scholar. Preparation and Incubation of Primary Hepatocytes—Hepatocytes were prepared from rats by collagenase perfusion by a modification (27Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5190) Google Scholar) of the method of Berry and Friend (28Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-520Crossref PubMed Scopus (3601) Google Scholar). The final wash of cells in Ca2+-free phosphate-buffered saline, pH 7.4, was performed at 37 °C in the presence of 1 mm l-carnitine 20 min prior to incubation, to compensate for carnitine lost from cells during preparative procedures (29Skorve J. Rustan A.C. Berge R.K. Lipids. 1995; 30: 987-994Crossref PubMed Scopus (6) Google Scholar, 30Tran T.N. Christophersen B.O. Biochim. Biophys. Acta. 2001; 1533: 255-265Crossref PubMed Scopus (12) Google Scholar). Production of acid-soluble products was measured using fatty acids as substrates. The assay mixture contained the following components in a total volume of 1.0 ml: 400 μm 1-14C-labeled fatty acid (0.25 μCi/ml) in Ca2+-free phosphate-buffered saline, pH 7.4, containing 137 mm NaCl, 2.7 mm KCl, 8 mm Na2HPO4, 1.5 mm KH2PO4, and 1% (w/v) fatty acid-free bovine serum albumin. Incubation was for 1 h at 30 °C in the presence of 2 × 106 cells. Acid-soluble products were measured essentially as given by Christiansen et al. (31Christiansen R. Borrebaek B. Bremer J. FEBS Lett. 1976; 62: 313-317Crossref PubMed Scopus (88) Google Scholar). Rates of oxygen uptake by cells were measured polarographically at 37 °C as given above for isolated mitochondria, except that the reaction mixture contained in a total volume of 1.0 ml: 400 μm fatty acid in Ca2+-free phosphate-buffered saline, pH 7.4, containing 1% fatty acid-free bovine serum albumin and 2 × 106 cells. The experiment was started with the addition of fatty acid substrate after a 10-min preincubation at 37 °C with an open measuring chamber. Where used, 5 μm FCCP was added near the end of each 10-min experiment. Rates of β-oxidation were measured as for isolated mitochondria (above) except that [1-14C]palmitic acid was used as substrate. Production of ketone bodies was assessed by measuring the amount of d-β-hydroxybutyrate (Sigma, kit number 310A) present in a neutralized, perchloric acid extract of the reaction mixture after completion of the polarographic experiment, and corrected for the contents of that component in unincubated cells. Measurement of Protein Motive Force—The proton electrochemical potential (Δp) were measured by recording the distribution across the mitochondrial inner membrane of labeled TTP as the permeant cation and labeled 5,5-dimetyloxazolidine-2,4-dione as the permeant weak acid, via centrifugation through an oil layer essentially as given by Dawson et al. (32Dawson A. Klingenberg M. Krämer R. Daley-Usmar V.W. Rickwood D. Wilson M.T. Mitochondria, A Practical Approach. IRL Press, Oxford, UK1987: 35-78Google Scholar). Briefly, the electrical potential difference (ΔΨ) was measured by incubating mitochondria in a medium containing the following concentrations of components: 150 mm KCl, 5 mm K-HEPES, pH 7.4, 2.5 mm Tris phosphate, 0.5 mm malate, 10 mm 5,5-dimetyloxazolidine-2,4-dione, 100 μm inulin, 10 μm TTP, 50 mg of dextran 40 (to facilitate passage of mitochondria though an oil layer (33LaNoue K.F. Walajtys E.I. Williamson J.R. J. Biol. Chem. 1973; 248: 7171-7183Abstract Full Text PDF PubMed Google Scholar)), 0.18 μCi/ml [14C]TTP, and 10 mg of mitochondrial protein in a total of 2.0 ml. Incubation was performed in uncapped tubes at 25 °C. For measurement of the transmembrane pH difference (ΔpH), labeled TTP was exchanged for 0.18 μCi of [14C]5,5-dimetyloxazolidine-2,4-dione/ml + 0.6 μCi of [3H]inulin/ml. Experiments were started by addition of substrate, either 40 nmol of palmitoyl-l-carnitine/mg of mitochondrial protein, in the presence of 1 mm malate, pH 7.4, or 2.5 mm Tris succinate, pH 7.4, in the presence of 10 μg of rotenone. At 3 and 5 min appropriate samples were withdrawn and added to tubes previously charged with a silicon oil layer (ρ = 1.5) above a layer of 10% (w/v) perchloric acid (ρ = 1.8), followed by centrifugation for 1 min at 15,000 rpm (Eppendorf microcentrifuge). For the ΔΨ experiments, the disappearance of [14C] from the upper, incubation medium layer was recorded by subjecting aliquots of that layer to scintillation counting, whereas for the ΔpH experiments, aliquots were withdrawn from both the upper as well from the bottom (perchloric acid) layers, and subjected to dual-channel scintillation counting. Determination of intra-mitochondrial volume was made by recording the transmembrane distributions of [14C]sucrose and 3H2O (32Dawson A. Klingenberg M. Krämer R. Daley-Usmar V.W. Rickwood D. Wilson M.T. Mitochondria, A Practical Approach. IRL Press, Oxford, UK1987: 35-78Google Scholar). The sucrose impermeable space of liver mitochondria isolated from animals given dietary palmitate was 1.27 ± 0.11 versus 1.28 ± 0.14 μl/mg of protein for the TTA-treated ones (n = 12). No correction was applied for possible overestimation of ΔΨ because of passive TTP binding since the measured ΔΨ was always higher than the figure where deviation from Nernst behavior has been demonstrated (34Warhurst I.W. An Investigation of the Anion Conducting Pore of Rat Liver Mitochondria. Doctoral dissertation, University of East Anglia, Norwich, United Kingdom1983Google Scholar). Parallel, polarographic incubations were used to verify that a steady state rate of oxygen uptake existed within the time frame of withdrawal of aliquots. Mitochondrial Fatty Acid Composition—TTA suspended in 0.5% carboxymethylcellulose was administered to rats by orogastric intubation (150 mg/kg body weight) once daily for 10 days. Control animals received carboxymethylcellulose only. PPAR–/– and PPAR+/+ mice were given the diet described above. Lipids were extracted from the liver mitochondrial fractions of rats and mice, transesterified with BF3-methanol, and analyzed essentially as described in Ref. 35Bjorndal B. Helleland C. Boe S.O. Gudbrandsen O.A. Kalland K.H. Bohov P. Berge R.K. Lillehaug J.R. J. Lipid Res. 2002; 43: 1630-1640Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar. The methyl esters of fatty acids were analyzed on a GC 8000 Top gas chromatograph (Carlo Erba Instrument), equipped with a flame ionization detector, programmable temperature of vaporization injector, AS 800 autosampler (Carlo Erba Instrument), and a capillary column (60 m × 0.25 mm) containing a highly polar SP 2340 phase with film thickness 0.20 mm (Supelco). Natural occurring fatty acids were positively identified by comparison to known standards (Larodan Fine Chemicals, Malmö, Sweden) and verified by mass spectrometry. Quantification of the fatty acids was based on heneicosanoic acid (21:0) as an internal standard. Isolation of mRNA and Quantitation by Real-time PCR Analysis— Total RNA was extracted from freeze-clamped liver using Trizol™ reagent (Invitrogen). Quantitative real-time PCR was carried out using ABI PRISM™ 7900 HT sequence detection system (Applied Biosystems, Foster City, CA) with conditions and reagents as recommended by the manufacturer. Each sample was analyzed in triplicate. Sequence-specific PCR primers and TaqMan probes for UCP-2 and the GAPDH cDNAs were designed using Primer Express software (Applied Biosystems). The following primers and probes were used: GAPDH: primers, 5′-TGCACCACCAACTGCTTAGC-3′ and 5′-CAGTCTTCTGAGTGGCAGTGATG-3′, and probe, 5′-TGGAAGGGCTCATGACCACAGTCCA-3′; UPC-2: primers, 5′-TGGCCTCTACGACTCTGTAAAGC-3′ and 5′-CAGGGCACCTGTGGTGCTA-3′, and probe, 5-CAAGGGCTCAGAGCATGCAGGCA-3′. The GAPDH was used as endogenous control for normalization of cDNA amounts. This analysis was also performed on isolated hepatocytes (performed as described above) that were purified by centrifugation on a 45% Percoll cushion to minimize the influence from Kuppfer cells (17Armstrong M.B. Towle H.C. Am. J. Physiol. 2001; 281: E1197-E1204PubMed Google Scholar). Western Analysis—Protein from extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane (Hybond ECL, Amersham) according to standard techniques. Blots were probed with a polyclonal goat antibody to UCP-2 (sc-6525, Santa Cruz Biotechnology Inc.) and goat horseradish peroxidase-conjugated anti-rabbit antibody (Bio-Rad). Statistics and Presentation of Results—The data are presented as mean ± S.D., and differences were evaluated by a two-sample Student's t test (two-tailed distribution) where relevant. p < 0.05 was regarded as statistical significant. Energy Parameters in Rat Liver—To investigate whether the hypolipidemia caused by supplementing rat diets with 3-thia fatty acids is associated with perturbation of the liver energy state, we established a 7-day regime of feeding animals TTA compared with a set of control animals receiving palmitic acid (which does not cause hypolipidemia). TTA treatment resulted in a lowering of energy state parameters such as phosphate potential by 30% and energy charge by 13%, compared with control livers (Table I). Simultaneously, the liver NAD+/NADH and NADP+/NADPH redox pairs became more oxidized. Whereas the total amounts of adenine nucleotides or inorganic phosphate remained unaffected by the dietary treatment, the amounts of nicotinamide adenine dinucleotide almost doubled and that of nicotinamide adenine dinucleotide phosphate increased by 30%, strongly suggesting that TTA stimulates the biosynthetic pathways for pyridine nucleotides in liver or, alternatively, inhibits glycohydrolases, which converts NAD to nicotinamide.Table IChanges in contents and composition of nucleotides in livers of rats given TTA versus palmitic acid as dietary supplementNucleotide componentsDietary supplementPalmitic acidTTAAMP + ADP + ATP3898 ± 343769 ± 131NSaNS, not significant for differences between TTA- and palmitic acid-treated rats; n = 5.GDP + GTP479 ± 34425 ± 17*bp < 0.05.NAD + NADH781 ± 361421 ± 101†cp < 0.01.NADP + NADPH338 ± 23457 ± 18*Inorganic phosphate3500 ± 6133678 ± 174NSTissue energy parameters[NAD]/[NADH]27.1 ± 1.2258.4 ± 4.3†[NADP]/[NADPH]0.40 ± 0.120.60 ± 0.06†Phosphorylation state497 ± 69340 ± 35†Energy charge0.73 ± 0.030.64 ± 0.06*a NS, not significant for differences between TTA- and palmitic acid-treated rats; n = 5.b p < 0.05.c p < 0.01. Open table in a new tab Oxidative Capacities of Primary Hepatocytes from TTA-treated Rats—Hepatocytes prepared from animals given TTA versus palmitic acid in their diets would be expected to expose facets of a mechanism for lowering the liver energy state. Accordingly, a study was undertaken of cellular production of acid-soluble products from labeled fatty acid substrates (as a measure of β-oxidation) as well as of fatty acid-stimulated ketogenesis and respiratory rates. As shown in Table II, TTA feeding caused increases in rates of cellular β-oxidation by 1.4–1.9-fold, eicosapentaenoic acid (EPA) being the better substrate among the fatty acids supplied. Stimulation by TTA feeding on ketogenesis in cells was more pronounced for palmitic acid (2-fold) and EPA (1.9-fold) than for docosahexaenoic acid (DHA; 1.5-fold) as sources of carbon. The fatty acid-stimulated rates of oxygen uptake responded similarly (1.3-fold increase) to TTA feeding of source animals. The oxygen uptake rates measured in the uncoupled state (preincubation with FCCP) were almost identical with any fatty acid substrate, regardless of feeding regime, indicating that the capacity of the mitochondrial respiratory chain had not been altered by the diet supplement. However, as a consequence there was some indication of a lowering of energy transducing activity in cells f" @default.
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